Methods and compositions for stimulating bone growth using nitric oxide releasing bisphosphonate conjugates (no-bisphosphonate)

ABSTRACT

The invention relates to compositions and methods for use in treating skeletal system disorders in a vertebrate at risk for bone loss, and in treating conditions that are characterized by the need for bone growth, in treating fractures, and in treating cartilage disorders. More specifically, the invention concerns the use of NO-bisphosphonate assembly for enhancing bone growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/428,309, filed May 2, 2003, now U.S. Pat. No. 7,288,535, which claimsthe benefit of U.S. Provisional Application Ser. No. 60/377,898, filedMay 2, 2002. The contents of each of these documents are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

The invention relates to compositions and methods for use in treatingskeletal system disorders in a vertebrate at risk for bone loss, and intreating conditions that are characterized by the need for bone growth,in treating fractures, and in treating cartilage disorders. Morespecifically, the invention concerns the use of NO-bisphosphonateactivity, e.g., a NO-bisphosphonate compound, for enhancing bone growth.

BACKGROUND OF THE INVENTION

Bone is subject to constant breakdown and re-synthesis in a complexprocess mediated by osteoblasts, which produce new bone, andosteoclasts, which destroy bone. The activities of these cells areregulated by a large number of cytokines and growth factors, many ofwhich have now been identified and cloned.

There is a plethora of conditions which are characterized by the need toenhance bone formation or to inhibit bone resorption. Perhaps the mostobvious is the case of bone fractures, where it would be desirable tostimulate bone growth and to hasten and complete bone repair. Agentsthat enhance bone formation would also be useful in facialreconstruction procedures. Other bone deficit conditions include bonesegmental defects, periodontal disease, metastatic bone disease,osteolytic bone disease and conditions where connective tissue repairwould be beneficial, such as healing or regeneration of cartilagedefects or injury. Also of great significance is the chronic conditionof osteoporosis, including age-related osteoporosis and osteoporosisassociated with post-menopausal hormone status. Other conditionscharacterized by the need for bone growth include primary and secondaryhyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis,and glucocorticoid-related osteoporosis.

Bone fractures are still treated exclusively using casts, braces,anchoring devices and other strictly mechanical means. Further bonedeterioration associated with post-menopausal osteoporosis has beentreated with estrogens or bisphosphonates, which may have drawbacks forsome individuals. Treatment of bone or other skeletal disorders, such asthose associated with cartilage, can be achieved either by enhancingbone formation or inhibiting bone resorption or both.

Bone tissue is an excellent source for factors which have the capacityfor stimulating bone cells. Thus, extracts of bovine bone tissueobtained from slaughterhouses contain not only structural proteins whichare responsible for maintaining the structural integrity of bone, butalso biologically active bone growth factors which can stimulate bonecells to proliferate. Among these latter factors are transforming growthfactor β, the heparin-binding growth factors (e.g., acidic and basicfibroblast growth factor), the insulin-like growth factors (e.g.,insulin-like growth factor I and insulin-like growth factor II), and arecently described family of proteins called bone morphogenetic proteins(BMPs). All of these growth factors have effects on other types ofcells, as well as on bone cells.

The cells which are responsible for forming bone are osteoblasts. Asosteoblasts differentiate from precursors to mature bone-forming cells,they express and secrete a number of enzymes and structural proteins ofthe bone matrix, including Type-1 collagen, osteocalcin, osteopontin andalkaline phosphatase. They also synthesize a number of growth regulatorypeptides which are stored in the bone matrix, and are presumablyresponsible for normal bone formation. These growth regulatory peptidesinclude the BMPs (Harris S., et al. (1994), supra). In studies ofprimary cultures of fetal rat calvarial osteoblasts, BMPs 1, 2, 3, 4,and 6 are expressed by cultured cells prior to the formation ofmineralized bone nodules (Harris S., et al. (1994), supra). Likealkaline phosphatase, osteocalcin and osteopontin, the BMPs areexpressed by cultured osteoblasts as they proliferate and differentiate.

These data are, however, contrary to reports that dexamethasone andother inducers, such as BMPs, induce osteoblastic differentiation andstimulate osteocalcin mRNA (Bellows, C. G., et al., Develop Biol (1990)140:132-38; Rickard, D. J., et al., Develop Biol (1994) 161:218-28). Inaddition, Ducy, P., et al., Nature (1996) 382:448-52 have recentlyreported that osteocalcin deficient mice exhibit a phenotype marked byincreased bone formation and bones of improved functional quality,without impairment of bone resorption. Ducy, et al., state that theirdata suggest that osteocalcin antagonists may be of therapeutic use inconjunction with estrogen replacement therapy (for prevention ortreatment of osteoporosis). However, there continues to be a need foradditional treatments to stimulate bone growth or to mitigate bone loss.

Bisphosphonates, formerly called diphosphonates are compoundscharacterized by two C—P bonds. If the two bonds are located on the samecarbon atom, resulting in a P—C—P structure, the compounds are calledgeminal bisphosphonates. They are therefore analogues of pyrophosphatethat contain a carbon instead of an oxygen. There are a number of knownpharmacologically active bisphosphonates including alendronate,Clodronate, etidronate, ibandronate, icadronate, pamidronate,risedronate, tiludronate and zoledronate. The main effect of thesepharmacologically active bisphosphonates is to inhibit resorption bothin vitro and in vivo. These effects are related to the marked affinityof these compounds for solid-phase calcium phosphate, on the surface ofbone to which they bond strongly. In essence they target bone and elicitthere pharmacological inhibition of osteoclast activity there. The modeof action of the bisphosphonates is still not completely elucidated.There is no doubt that their action in vivo is mediated mostly, if notcompletely, through mechanisms other than the physiochemical inhibitionof crystal dissolution. There is a general consensus that thebisphosphonates act by inhibiting the activity of osteoclasts.Osteoclasts are inhibited when they come into contact withbisphosphonates-containing bone. This supports the hypothesis thatbisphosphonates are deposited onto bone because of their strong affinityfor the mineral, and that the osteoclasts are then inhibited when theystart to engulf bisphosphonates-containing bone. The biochemicalmechanisms by which bisphosphonates inhibit osteoclast activity arestill unclear and may well be that more than one mechanism is operating.

The bisphosphonates investigated up to now appear to be absorbed,stored, and excreted unaltered in the body. Thus, bisphosphonates seemto be non-biodegradable, both in animals and in solution. Most of thepharmacokinetics data on the bisphosphonates have been obtained withetidronate, clodronate and pamidronate. The intestinal absorption liesbetween 1% and 10%. Between 20% and 50% of the absorbed bisphosphonateis localized to the bone, the remainder being rapidly excreted in theurine.

Although the nitrogen-containing bisphosphonates such as alendronate andpamidronate, have been shown to be effective in preventing bone loss,these drugs also appear capable of causing injury to the uppergastrointestinal tract, and there have been several case reports ofsevere esophagitis in patients treated with alendronate. Alendronate hasalso been shown to cause erosions and ulcers in the human stomach and tointerfere with the healing of pre-existing lesions when given to healthyvolunteers at doses that are prescribed for the treatment ofosteoporosis and Pagets disease of bone.

The half-life of circulating bisphosphonates is short, in the rat onlyon the order of minutes. In man it is somewhat longer, about 2 hours.Acute, subacute, and chronic administration in several animal specieshave revealed little toxicity. Teratogenic, mitogenic and carcinogenictests have been negative.

Other agents appear to operate by preventing the resorption of bone.Thus, U.S. Pat. No. 5,280,040 discloses compounds described as useful inthe treatment of osteoporosis. These compounds putatively achieve thisresult by preventing bone resorption.

Nitric oxide (NO) has recently been shown to have profound effects onthe metabolic activity of bone. This biologically active molecule isgenerated biologically by a set of enzymes called nitric oxidesynthases. Three known forms of the enzyme exist. Firstly, iNOS which isthe inducible form where the expression of the enzyme and therefore theproduction of nitric oxide can be induced by a number of inflammatorystimuli. Secondly eNOS which is the constitutive form which cannot beinduced.

Endothelial dysfunction defined as the impaired ability of vascularendothelium to stimulate vasodilation plays a key role in thedevelopment of atherosclerosis and in various pathological conditionswhich predispose a subject to atherosclerosis, such ashypercholesterolemia, hypertension, type 2 diabetes,hyperhomocyst(e)inemia and chronic renal failure. The major cause of theendothelial dysfunction is decreased bioavailability of nitric oxide(NO), a potent biological vasodilator produced in vascular endotheliumfrom L-arginine by the endothelial NO synthase (eNOS). In vasculardiseases, the bioavailability of NO can be impaired by variousmechanisms, including decreased NO production by eNOS, and/or enhancedNO breakdown due to increased oxidative stress. The deactivation of eNOSis often associated with elevated plasma levels of its endogenousinhibitor, N(G),N(G)-dimethyl-L-arginine (ADMA). Inhypercholesterolemia, a systemic deficit of NO may also increase thelevels of low density lipoproteins (LDL) by modulating its synthesis andmetabolism by the liver, as suggested by recent in vivo and in vitrostudies using organic NO donors. Therapeutic strategies aiming to reducethe risk of vascular diseases by increasing bioavailability of NOcontinue to be developed.

Nitric oxide (NO) is a free radical which has important effects on bonecell function. The endothelial isoform of nitric oxide synthase (eNOS)is widely expressed in bone on a constitutive basis, whereas inducibleNOS is only expressed in response to inflammatory stimuli. It iscurrently unclear whether neuronal NOS is expressed by bone cells.Pro-inflammatory cytokines such as IL-1 and TNF cause activation of theiNOS pathway in bone cells and NO derived from this pathway potentiatescytokine and inflammation induced bone loss. These actions of NO arerelevant to the pathogenesis of osteoporosis in inflammatory diseasessuch as rheumatoid arthritis, which are characterized by increased NOproduction and cytokine activation. Interferon gamma is a particularlypotent stimulator of NO production when combined with other cytokines,causing very high concentrations of NO to be produced. These high levelsof NO inhibit bone resorption and formation and may act to suppress boneturnover in severe inflammation. The eNOS isoform seems to play a keyrole in regulating osteoblast activity and bone formation since eNOSknockout mice have osteoporosis due to defective bone formation. Otherstudies have indicated that the NO derived from the eNOS pathway acts asa mediator of the effects of estrogen in bone. eNOS also mediates theeffects of mechanical loading on the skeleton where it acts along withprostaglandins, to promote bone formation and suppress bone resorption.Pharmacological NO donors have been shown to increase bone mass inexperimental animals and preliminary evidence suggests that these agentsmay also influence bone turnover in man. These data indicate that theL-arginine/NO pathway represents a novel target for therapeuticintervention in the prevention and treatment of bone diseases.

Because of the importance of nitric oxide in many biological eventsnumerous NO releasing compounds are now being synthesized. Many of theseinvolve the use of linking NO to non-steroidal anti-inflammatory drugs(NSAIDS) such as flurbiprofen, ketoprofen, diclofenac and naproxen.These agent have been shown to spare the GI-tract from the undesiredeffects of NSAIDs by increasing blood flow and mucus secretion as wellas reducing free radical generation in the stomach.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a method to enhancebone formation or bone mineral density, or to treat a pathological bonecondition or to treat a degenerative joint condition in a vertebratesubject, which method comprises administering to a vertebrate subject inneed of such treatment an effective amount of a bisphosphonate to whichis attached nitric oxide in a form which can be released(NO-bisphosphonate), whereby bone formation or bone mineral density isenhanced, or said pathological bone condition or degenerative jointcondition is treated, in said vertebrate subject. The NO-bisphosphonatecan be used alone, in combinations, or can be used in conjunction withan additional or secondary agent that promotes bone growth or inhibitsbone resorption.

In another aspect, the present invention is directed to a combination,preferably in the form of a pharmaceutical composition, whichcombination comprises a NO-bisphosphonate compound and a nonNO-bisphosphonate compound that promotes bone growth or inhibits boneresorption. Kits and articles of manufactures comprising aNO-bisphosphonate compound, whether alone, in combinations, or with anon-NO-bisphosphonate compound that promotes bone growth or inhibitsbone resorption, also are provided.

Any suitable bone enhancer or bone resorption inhibitor can be used inthe combination. Exemplary of such agents that can be used in thecombination include bone morphogenetic factors, anti-resorptive agents,osteogenic factors, cartilage-derived morphogenetic proteins, growthhormones, estrogens, bisphosphonates, statin, differentiating factors,compounds that inhibit activity of NF-κB, e.g., anti-NF-κB antibodies,compounds that inhibit production of NF-κB, e.g., anti-NF-κB antisenseoligos, compounds that inhibit activity of proteasomal activity, e.g.,antibodies that specifically bind to proteasomal proteins, and compoundsthat inhibit production of a proteasome protein, e.g., antisense oligosthat are complementary to genes or RNAs that encode proteasomalproteins. For clinical uses, the antibodies are preferably monoclonal orhumanized antibodies.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided methods oftreating bone defects (including osteoporosis, fractures, osteolyticlesions and bone segmental defects) in subjects suffering therefrom,said method comprising administering to said subject, in an amountsufficient to stimulate bone growth, a NO-bisphosphonate.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications and sequences from GenBankand other databases referred to herein are incorporated by reference intheir entirety. If a definition set forth in this section is contrary toor otherwise inconsistent with a definition set forth in applications,published applications and other publications and sequences from GenBankand other data bases that are herein incorporated by reference, thedefinition set forth in this section prevails.

As used herein, “a” or “an” means “at least one” or “one or more.” Asused herein, “or” means in the alternative as well as in combination.

As used herein, “enhance” means to promote, increase, or stimulate boneformation or growth, or bone mineral density, in a vertebrate animal.

As used herein, “an effective amount” of a compound for treating aparticular disease is an amount that is sufficient to ameliorate, or insome manner reduce the symptoms associated with the disease. Such amountmay be administered as a single dosage or may be administered accordingto a regimen, whereby it is effective. The amount may cure the diseasebut, typically, is administered in order to ameliorate the symptoms ofthe disease. Repeated administration may be required to achieve thedesired amelioration of symptoms. Within the present invention, an“effective amount” of a composition is that amount which produces astatistically significant effect. For example, an “effective amount” fortherapeutic uses is the amount of the composition comprising an activecompound herein required to provide a clinically significant increase inhealing rates in fracture repair; reversal of bone loss in osteoporosis;reversal of cartilage defects or disorders; prevention or delay of onsetof osteoporosis; stimulation and/or augmentation of bone formation infracture non-unions and distraction osteogenesis; increase and/oracceleration of bone growth into prosthetic devices; and repair ofdental defects. Such effective amounts will be determined using routineoptimization techniques and are dependent on the particular condition tobe treated, the condition of the patient, the route of administration,the formulation, and the judgment of the practitioner and other factorsevident to those skilled in the art.

As used herein, “pharmaceutically acceptable salts, esters or otherderivatives” include any salts, esters or derivatives that may bereadily prepared by those of skill in this art using known methods forsuch derivatization and that produce compounds that may be administeredto animals or humans without substantial toxic effects and that eitherare pharmaceutically active or are prodrugs.

As used herein, “treatment” means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Treatment also encompasses any pharmaceutical use of thecompositions herein.

As used herein, “amelioration” of the symptoms of a particular disorderby administration of a particular pharmaceutical composition refers toany lessening, whether permanent or temporary, lasting or transient thatcan be attributed to or associated with administration of thecomposition.

As used herein, “substantially pure” means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers or isomers. In such instances, furtherpurification might increase the specific activity of the compound.

As used herein, “antibody” includes antibody fragments, such as Fabfragments, which are composed of a light chain and the variable regionof a heavy chain.

As used herein, “humanized antibodies” refer to antibodies that aremodified to include “human” sequences of amino acids so thatadministration to a human will not provoke an immune response. Methodsfor preparation of such antibodies are known. For example, the hybridomathat expresses the monoclonal antibody is altered by recombinant DNAtechniques to express an antibody in which the amino acid composition ofthe non-variable regions is based on human antibodies. Computer programshave been designed to identify such regions.

The term “substantially” identical or homologous or similar varies withthe context as understood by those skilled in the relevant art andgenerally means at least 70%, preferably means at least 80%, morepreferably at least 90%, and most preferably at least 95% identity.

As used herein, a “composition” refers to any mixture. It may be asolution, a suspension, liquid, powder, paste, aqueous, non-aqueous orany combination thereof.

As used herein, a “combination” refers to any association between two oramong more items.

As employed herein, the term “subject” embraces human as well as otheranimal vertebrate species, such as, for example, canine, feline, bovine,porcine, rodent, and the like. It will be understood by the skilledpractitioner that the subject is one appropriate to the desirability ofenhancing bone formation or bone mineral density. Preferably the subjectis a mammal, more preferably a primate, and most preferably a human.

As used herein, “treat” or “treatment,” as related to bone growthdefects, include a postponement of development of bone deficit symptomsand/or a reduction in the severity of such symptoms that will or areexpected to develop. These terms further include ameliorating existingbone or cartilage deficit symptoms, preventing additional symptoms,ameliorating or preventing the underlying metabolic causes of symptoms,preventing or reversing bone resorption and/or encouraging bone growth.Thus, the terms denote that a beneficial result has been conferred on avertebrate subject with a cartilage, bone or skeletal deficit, or withthe potential to develop such deficit.

By “bone deficit” is meant an imbalance in the ratio of bone formationto bone resorption, such that, if unmodified, the subject will exhibitless bone than desirable, or the subject's bones will be less intact andcoherent than desired. Bone deficit may also result from fracture, fromsurgical intervention or from dental or periodontal disease. By“cartilage defect” is meant damaged cartilage, less cartilage thandesired, or cartilage that is less intact and coherent than desired.“Bone disorders” includes both bone deficits and cartilage defects.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

B. METHODS FOR ENHANCING BONE FORMATION AND USES THEREOF

In one aspect, the present invention is directed to a method to enhancebone formation or bone mineral density, or to treat a pathological bonecondition or to treat a degenerative joint condition in a vertebratesubject, which method comprises administering to a vertebrate subject inneed of such treatment an effective amount of a NO-bisphosphonate,whereby bone formation or bone mineral density is enhanced, or saidpathological bone condition or degenerative joint condition is treated,in said vertebrate subject. The compound can be used alone or can beused in conjunction with an additional or secondary agent that promotesbone growth or inhibits bone resorption.

Any NO-bisphosphonate can be used in the present method. In particularthis includes specific NO-bisphosphonates such asNO-(4-amino-1-hydroxybutylidene)bis-phosphonate (NO-alendronate),NO-(3-Amino-1-hyroxypropylidene)bis-phosphonate (NO-pamidronate),NO-(Dichloromethylene)-bis-phosphonate, (NO-clodronate),NO-[1-Hydroxy-2(3-pyridinyl)-ethylidene]-bis-phosphonate(NO-risedronate),NO-[1-Hyroxy-2-(1H-imidazole-1-yl)ethylidene]-bis-phosphonate(NO-zolondronate), NO-(1-Hydroxyethylidene)-bis-phosphonate(NO-etidronate),NO-[1-Hydroxy-3-(methylpentylamino)propylidene]-bis-phosphonate(NO-ibandronate), NO-[[(4-Clorophenyl)thio]methylene]-bis-phosphonate(NO-tiludronate), NO-(6-Amino-1-hydroxyhexylidene)-bis-phosphonate(NO-neridronate),NO-([3-(Dimethylamino)-1-hydroxypropylidene]-bis-phosphonate(NO-olpadronate), NO-[(Cycloheptylamino)-methylene]-bis-phosphonate(NO-icadronate) andNO-[1-Hydroxy-2-imidazo-(1,2-a)pyridine-3-ylethylidene]-bis-phosphonate(NO-YH529).

The present method can be used for treating any diseases, disorders orconditions that are associated with bone formation defects, whethercaused by defective bone growth, over-active bone resorption or both.Any pathological dental conditions or degenerative joint conditions canbe treated with the present method. Exemplary conditions that can betreated by the present method include osteoporosis, bone fracture ordeficiency, bone segmental defects, primary or secondaryhyperparathyroidism, periodontal disease or defect, metastatic bonedisease, osteolytic bone disease, post-plastic surgery, post-prostheticjoint surgery, and post-dental implantation.

Other uses of the present method include, but are not limited to, repairof bone defects and deficiencies, such as those occurring in closed,open and non-union fractures; prophylactic use in closed and openfracture reduction; promotion of bone healing in plastic surgery;stimulation of bone in-growth into non-cemented prosthetic joints anddental implants; elevation of peak bone mass in pre-menopausal women;treatment of growth deficiencies; treatment of periodontal disease anddefects, and other tooth repair processes; increase in bone formationduring distraction osteogenesis; and treatment of other skeletaldisorders, such as age-related osteoporosis, post-menopausalosteoporosis, diabetes-related osteoporosis, glucocorticoid-induced orrelated osteoporosis, or disuse osteoporosis and arthritis, or anycondition that benefits from stimulation of bone formation; repair ofcongenital, trauma-induced or surgical resection of bone (for instance,for cancer treatment) and in cosmetic surgery; limiting or treatingcartilage defects, injuries or disorders; and may be useful in woundhealing or tissue repair.

C. COMBINATIONS, KITS AND ARTICLES OF MANUFACTURE

In a specific embodiment, the present method can further compriseadministering to the subject an additional agent that promotes bonegrowth or inhibits bone resorption. The NO-bisphosphonate and thesecondary agent can be administered simultaneously or sequentially.

Any suitable bone enhancer or bone resorption inhibitor can be used inthis combination therapy as the additional agent. Exemplary compoundsthat can be used in the combination therapy include bone morphogeneticfactors, anti-resorptive agents, osteogenic factors, cartilage-derivedmorphogenetic proteins, growth hormones, estrogens, bisphosphonates,statin, differentiating factors, compounds that inhibit activity ofNF-κB, compounds that inhibit production of NF-κB, compounds thatinhibit activity of proteasomal activity and compounds that inhibitsproduction of a proteasome protein. Preferably, these compounds used forenhancing bone formation or treating pathological dental conditions ordegenerative joint conditions are disclosed below and those that aredisclosed in the following published PCT International PatentApplications also can be used: PCT/US 00/41360, filed Oct. 20, 2000; andWO 00/02548.

Small molecules which are able to stimulate bone formation have beendisclosed in PCT applications WO98/17267 published 30 Apr. 1998,WO97/15308 published 1 May 1997 and WO97/48694 published 24 Dec. 1997.These agents generally comprise two aromatic systems spatially separatedby a linker. In addition, PCT application WO98/25460 published 18 Jun.1998, discloses the use of the class of compounds known as statins inenhancing bone formation.

The NO-bisphosphonate and additional agent can be formulated in a singlepharmaceutical composition. Alternatively, they can be formulated asseparate pharmaceutical compositions.

Other known inhibitors of proteasomal activity, NF-κB or both can beascertained from the literature or compounds can be tested for theseactivities using assays known in the art. In addition, inhibitors, e.g.,antisense polynucleotides, which lower the level of effective expressionof the nucleotide sequence encoding the enzymes that have proteasomalactivity or of the nucleotide sequence encoding NF-κB can be assessedand used in the invention methods. Also provided are compounds such assulfasalazine (Liptay, et al., Br. J. Pharmacol., 128(7):1361-9 (1999));and Wahl, et al., J. Clin. Invest., 101(5):1163-74 (1998)) and calpaininhibitor II.

E. ASSAYS FOR NO-BISPHOSPHONATE

Numerous assays can be used to identify and/or assess the efficacy ofcompounds that can be used in the present methods and combinations orpharmaceutical compositions.

The production of nitric oxide in cell culture media, serum or plasmacan be measured by using available assay kits for total nitric oxide(R&D systems) as well as Nitrite/Nitrate (NO₂ ⁻/NO₃ ⁻) kits (R&Dsystems). Intracellular activation of soluble guanylate cyclase bynitric oxide can be measured by the use of cyclic AMP/GMP enzymeimmunoassay kits (Cayman, Ann Arbor).

Screening Assays—Bone

The osteogenic activity of the compounds used in the methods of theinvention can be verified using in vitro screening techniques, such asthe assessment of transcription of a reporter gene coupled to a bonemorphogenetic protein-associated promoter or in alternative assays.

Techniques for ABA Screening Assay

A rapid throughput screening test for compounds that stimulate boneformation by demonstration that they are capable of stimulatingexpression of a reporter gene linked to a BMP promoter (a surrogate forthe production of bone morphogenetic factors that are endogenouslyproduced) is described in U.S. application Ser. No. 08/458,434, filed 2Jun. 1995, the entire contents of which are incorporated herein byreference. This assay is also described as a portion of a study ofimmortalized murine osteoblasts (derived from a mouse expressing atransgene composed of a BMP2 promoter driving expression of T-antigen)in Ghosh-Choudhery, N., et al., Endocrinology (1996) 137:331-39. In thisstudy, the immortalized cells were stably transfected with a plasmidcontaining a luciferase reporter gene driven by a mouse BMP2 promoter(−2736/114 bp), and responded in a dose-dependent manner to recombinanthuman BMP2.

Briefly, the assay utilizes cells transformed permanently or transientlywith constructs in which the promoter of a bone morphogenetic protein,specifically BMP2 or BMP4, is coupled to a reporter gene, typicallyluciferase. These transformed cells are then evaluated for theproduction of the reporter gene product; compounds that activate the BMPpromoter will drive production of the reporter protein, which can bereadily assayed. Many thousands of compounds have been subjected to thisrapid screening technique, and only a very small percentage are able toelicit a level of expression of reporter gene 5-fold greater than thatproduced by vehicle. Compounds that activate the BMP promoter fall intogroups, where members of each group share certain structuralcharacteristics not present in inactive compounds. The active compounds(“BMP promoter-active compounds” or “active compounds”) are useful inpromoting bone or cartilage growth, and thus in the treatment ofvertebrates in need of bone or cartilage growth.

BMP promoter-active compounds can be examined in a variety of otherassays that test specificity and toxicity. For instance, non-BMPpromoters or response elements can be linked to a reporter gene andinserted into an appropriate host cell. Cytotoxicity can be determinedby visual or microscopic examination of BMP promoter- and/or non-BMPpromoter-reporter gene-containing cells, for instance. Alternatively,nucleic acid and/or protein synthesis by the cells can be monitored. Forin vivo assays, tissues may be removed and examined visually ormicroscopically, and optionally examined in conjunction with dyes orstains that facilitate histologic examination. In assessing in vivoassay results, it may also be useful to examine biodistribution of thetest compound, using conventional medicinal chemistry/animal modeltechniques.

Techniques for Neonatal Mouse Calvaria Assay (In Vitro)

An assay for bone resorption or bone formation is similar to thatdescribed by Gowen M. & Mundy G., J Immunol (1986) 136:2478-82. Briefly,four days after birth, the front and parietal bones of ICR Swiss whitemouse pups are removed by microdissection and split along the sagittalsuture. In an assay for resorption, the bones are incubated in BGJbmedium (Irvine Scientific, Santa Ana, Calif.) plus 0.02% (or lowerconcentration) β-methylcyclodextrin, wherein the medium also containstest or control substances. The medium used when the assay is conductedto assess bone formation is Fitton and Jackson Modified BGJ Medium(Sigma) supplemented with 6 ug/ml insulin, 6 ug/ml transferrin, 6 ng/mlselenous acid, calcium and phosphate concentrations of 1.25 and 3.0 mM,respectively, and ascorbic acid to a concentration of 100 ug/ml is addedevery two days. The incubation is conducted at 37° C. in a humidifiedatmosphere of 5% CO₂ and 95% air for 96 hours.

These neonatal murine calvaria do contain measurable levels of alkalinephosphatase and this can be measured either from the calvaria itself orto some degree in the incubation media as a semi-quantitativemeasurement of bone formation activity. For measurement of alkalinephosphatase activity in the media, 50 ul is removed at the end of theassay. The activity is then measured using standard alkaline phosphatasereagents.

The bones are removed from the incubation media and fixed in 10%buffered formalin for 24-48 hours, decalcified in 14% EDTA for 1 week,processed through graded alcohols; and embedded in paraffin wax. Threeμm sections of the calvaria are prepared. Representative sections areselected for histomorphometric assessment of bone formation or boneresorption. Bone changes are measured on sections cut 200 μm apart.Osteoblasts and osteoclasts are identified by their distinctivemorphology.

Other auxiliary assays can be used as controls to determine non-BMPpromoter-mediated effects of test compounds. For example, mitogenicactivity can be measured using screening assays featuring aserum-response element (SRE) as a promoter and a luciferase reportergene. More specifically, these screening assays can detect signalingthrough SRE-mediated pathways, such as the protein kinase C pathway. Forinstance, an osteoblast activator SRE-luciferase screen and an insulinmimetic SRE-luciferase screen are useful for this purpose. Similarly,test compound stimulation of cAMP response element (CRE)-mediatedpathways can also be assayed. For instance, cells transfected withreceptors for PTH and calcitonin (two bone-active agents) can be used inCRE-luciferase screens to detect elevated cAMP levels. Thus, the BMPpromoter specificity of a test compound can be examined through use ofthese types of auxiliary assays.

F. FORMULATIONS AND ADMINISTRATIONS

The NO-bisphosphonate, whether alone or in combination with anadditional agent that promotes bone, may be administered systemically orlocally. For systemic use, the compounds herein are formulated forparenteral (e.g., intravenous, subcutaneous, intramuscular,intraperitoneal, intranasal or transdermal) or enteral (e.g., oral orrectal) delivery according to conventional methods. Intravenousadministration can be by a series of injections or by continuousinfusion over an extended period. Administration by injection or otherroutes of discretely spaced administration can be performed at intervalsranging from weekly to once to three times daily. Alternatively, thecompounds disclosed herein may be administered in a cyclical manner(administration of disclosed compound; followed by no administration;followed by administration of disclosed compound, and the like).Treatment will continue until the desired outcome is achieved. Ingeneral, pharmaceutical formulations will include a compound of thepresent invention in combination with a pharmaceutically acceptablevehicle, such as saline, buffered saline, 5% dextrose in water,borate-buffered saline containing trace metals or the like. Formulationsmay further include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces,lubricants, fillers, stabilizers, etc. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington'sPharmaceutical Sciences, latest edition, Mack Publishing Co., EastonPa., which is incorporated herein by reference. Pharmaceuticalcompositions for use within the present invention can be in the form ofsterile, non-pyrogenic liquid solutions or suspensions, coated capsules,suppositories, lyophilized powders, transdermal patches or other formsknown in the art. Local administration may be by injection at the siteof injury or defect, or by insertion or attachment of a solid carrier atthe site, or by direct, topical application of a viscous liquid, or thelike. For local administration, the delivery vehicle preferably providesa matrix for the growing bone or cartilage, and more preferably is avehicle that can be absorbed by the subject without adverse effects.

Delivery of compounds herein to wound sites may be enhanced by the useof controlled-release compositions, such as those described in PCTpublication WO 93/20859, which is incorporated herein by reference.Films of this type are particularly useful as coatings for prostheticdevices and surgical implants. The films may, for example, be wrappedaround the outer surfaces of surgical screws, rods, pins, plates and thelike. Implantable devices of this type are routinely used in orthopedicsurgery. The films can also be used to coat bone filling materials, suchas hydroxyapatite blocks, demineralized bone matrix plugs, collagenmatrices and the like. In general, a film or device as described hereinis applied to the bone at the fracture site. Application is generally byimplantation into the bone or attachment to the surface using standardsurgical procedures.

In addition to the copolymers and carriers noted above, thebiodegradable films and matrices may include other active or inertcomponents. Of particular interest are those agents that promote tissuegrowth or infiltration, such as growth factors. Exemplary growth factorsfor this purpose include epidermal growth factor (EGF), fibroblastgrowth factor (FGF), platelet-derived growth factor (PDGF), transforminggrowth factors (TGFs), parathyroid hormone (PTH), leukemia inhibitoryfactor (LIF), insulin-like growth factors (IGFs) and the like. Agentsthat promote bone growth, such as bone morphogenetic proteins (U.S. Pat.No. 4,761,471; PCT Publication WO90/11366), osteogenin (Sampath, et al.,Proc. Natl. Acad. Sci. USA (1987) 84:7109-13) and NaF (Tencer, et al.,J. Biomed. Mat. Res. (1989) 23: 571-89) are also preferred.Biodegradable films or matrices include calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyanhydrides, bone ordermal collagen, pure proteins, extracellular matrix components and thelike and combinations thereof. Such biodegradable materials may be usedin combination with non-biodegradable materials, to provide desiredmechanical, cosmetic or tissue or matrix interface properties.

Alternative methods for delivery of compounds of the present inventioninclude use of ALZET™ osmotic minipumps (Alza Corp., Palo Alto, Calif.);sustained release matrix materials such as those disclosed in Wang, etal. (PCT Publication WO 90/11366); electrically charged dextran beads,as disclosed in Bao, et al. (PCT Publication WO 92/03125);collagen-based delivery systems, for example, as disclosed in Ksander,et al., Ann. Surg. (1990) 211(3):288-94; methylcellulose gel systems, asdisclosed in Beck, et al., J. Bone Min. Res. (1991) 6(11):1257-65;alginate-based systems, as disclosed in Edelman, et al., Biomaterials(1991) 12:619-26 and the like. Other methods well known in the art forsustained local delivery in bone include porous coated metal prosthesesthat can be impregnated and solid plastic rods with therapeuticcompositions incorporated within them.

The compounds of the present invention may also be used in conjunctionwith agents that inhibit bone resorption. Antiresorptive agents, such asestrogen, bisphosphonates and calcitonin, are preferred for thispurpose. More specifically, the compounds disclosed herein may beadministered for a period of time (for instance, months to years)sufficient to obtain correction of a bone deficit condition. Once thebone deficit condition has been corrected, the vertebrate can beadministered an anti-resorptive compound to maintain the corrected bonecondition. Alternatively, the compounds disclosed herein may beadministered with an anti-resorptive compound in a cyclical manner(administration of disclosed compound, followed by anti-resorptive,followed by disclosed compound, and the like).

In additional formulations, conventional preparations such as thosedescribed below may be used.

Aqueous suspensions may contain the active ingredient in admixture withpharmacologically acceptable excipients, comprising suspending agents,such as methyl cellulose; and wetting agents, such as lecithin,lysolecithin or long-chain fatty alcohols. The said aqueous suspensionsmay also contain preservatives, coloring agents, flavoring agents,sweetening agents and the like in accordance with industry standards.

Preparations for topical and local application comprise aerosol sprays,lotions, gels and ointments in pharmaceutically appropriate vehicleswhich may comprise lower aliphatic alcohols, polyglycols such asglycerol, polyethylene glycol, esters of fatty acids, oils and fats, andsilicones. The preparations may further comprise antioxidants, such asascorbic acid or tocopherol, and preservatives, such as p-hydroxybenzoicacid esters.

Parenteral preparations comprise particularly sterile or sterilizedproducts. Injectable compositions may be provided containing the activecompound and any of the well known injectable carriers. These maycontain salts for regulating the osmotic pressure.

If desired, the osteogenic agents can be incorporated into liposomes byany of the reported methods of preparing liposomes for use in treatingvarious pathogenic conditions. The present compositions may utilize thecompounds noted above incorporated in liposomes in order to direct thesecompounds to macrophages, monocytes, as well as other cells and tissuesand organs which take up the liposomal composition. Theliposome-incorporated compounds of the invention can be utilized byparenteral administration, to allow for the efficacious use of lowerdoses of the compounds. Ligands may also be incorporated to furtherfocus the specificity of the liposomes.

Suitable conventional methods of liposome preparation include, but arenot limited to, those disclosed by Bangham, A. D., et al., J Mol Biol(1965) 23:238-252, Olson, F., et al., Biochim Biophys Acta (1979)557:9-23, Szoka, F., et al., Proc Natl Acad Sci USA (1978) 75:4194-4198,Kim, S., et al., Biochim Biophys Acta (1983) 728:339:348, and Mayer, etal., Biochim Biophys Acta (1986) 858:161-168.

The liposomes may be made from the present compounds in combination withany of the conventional synthetic or natural phospholipid liposomematerials including phospholipids from natural sources such as egg,plant or animal sources such as phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin,phosphatidylserine, or phosphatidylinositol and the like. Syntheticphospholipids that may also be used, include, but are not limited to:dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidycholine, and thecorresponding synthetic phosphatidylethanolamines andphosphatidylglycerols. Cholesterol or other sterols, cholesterolhemisuccinate, glycolipids, cerebrosides, fatty acids, gangliosides,sphingolipids, 1,2-bis(oleoyloxy)-3-(trimethyl ammonio) propane (DOTAP),N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA), andother cationic lipids may be incorporated into the liposomes, as isknown to those skilled in the art. The relative amounts of phospholipidand additives used in the liposomes may be varied if desired. Thepreferred ranges are from about 60 to 90 mole percent of thephospholipid; cholesterol, cholesterol hemisuccinate, fatty acids orcationic lipids may be used in amounts ranging from 0 to 50 molepercent. The amounts of the present compounds incorporated into thelipid layer of liposomes can be varied with the concentration of thelipids ranging from about 0.01 to about 50 mole percent.

The liposomes with the above formulations may be made still morespecific for their intended targets with the incorporation of monoclonalantibodies or other ligands specific for a target. For example,monoclonal antibodies to the BMP receptor may be incorporated into theliposome by linkage to phosphatidylethanolamine (PE) incorporated intothe liposome by the method of Leserman, L., et al., Nature (1980)288:602-604.

The compounds of the present invention may be used to stimulate growthof bone-forming cells or their precursors, or to induce differentiationof bone-forming cell precursors, either in vitro or ex vivo. Thecompounds described herein may also modify a target tissue or organenvironment, so as to attract bone-forming cells to an environment inneed of such cells. As used herein, the term “precursor cell” refers toa cell that is committed to a differentiation pathway, but thatgenerally does not express markers or function as a mature, fullydifferentiated cell. As used herein, the term “mesenchymal cells” or“mesenchymal stem cells” refers to pluripotent progenitor cells that arecapable of dividing many times, and whose progeny will give rise toskeletal tissues, including cartilage, bone, tendon, ligament, marrowstroma and connective tissue (see A. Caplan, J. Orthop. Res. (1991)9:641-50). As used herein, the term “osteogenic cells” includesosteoblasts and osteoblast precursor cells. More particularly, thedisclosed compounds are useful for stimulating a cell populationcontaining marrow mesenchymal cells, thereby increasing the number ofosteogenic cells in that cell population. In a preferred method,hematopoietic cells are removed from the cell population, either beforeor after stimulation with the disclosed compounds. Through practice ofsuch methods, osteogenic cells may be expanded. The expanded osteogeniccells can be infused (or reinfused) into a vertebrate subject in needthereof. For instance, a subject's own mesenchymal stem cells can beexposed to compounds of the present invention ex vivo, and the resultantosteogenic cells could be infused or directed to a desired site withinthe subject, where further proliferation and/or differentiation of theosteogenic cells can occur without immunorejection. Alternatively, thecell population exposed to the disclosed compounds may be immortalizedhuman fetal osteoblastic or osteogenic cells. If such cells are infusedor implanted in a vertebrate subject, it may be advantageous to“immunoprotect” these non-self cells, or to immunosuppress (preferablylocally) the recipient to enhance transplantation and bone or cartilagerepair.

The dosage required for the NO-bisphosphonate, whether alone or incombination with an additional agent that promotes bone (for example, inosteoporosis where an increase in bone formation is desired) ismanifested as a statistically significant difference in bone massbetween treatment and control groups. This difference in bone mass maybe seen, for example, as a 5-20% or more increase in bone mass in thetreatment group. Other measurements of clinically significant increasesin healing may include, for example, tests for breaking strength andtension, breaking strength and torsion, 4-point bending, increasedconnectivity in bone biopsies and other biomechanical tests well knownto those skilled in the art. General guidance for treatment regimens isobtained from experiments carried out in animal models of the disease ofinterest.

The dosage of the NO-bisphosphonate, whether alone or in combinationwith the secondary agent that promotes bone, will vary according to theextent and severity of the need for treatment, the activity of theadministered compound, the general health of the subject, and otherconsiderations well known to the skilled artisan. Generally, they can beadministered to a typical human on a daily basis as an oral dose ofabout 0.1 mg/kg-1000 mg/kg, and more preferably from about 1 mg/kg toabout 200 mg/kg. The parenteral dose will appropriately be 20-100% ofthe oral dose. While oral administration may be preferable in mostinstances where the condition is a bone deficit (for reasons of ease,patient acceptability, and the like), alternative methods ofadministration may be appropriate for selected compounds and selecteddefects or diseases. The compound levels can be monitored by anysuitable methods known in the art (See, e.g., Bouley et al., Ther. Drug.Monit., 23(1):56-60 (2001); and Langmann et al., J. Chromatogr. B.Biomed. Sci. Appl., 735(1):41-50 (1999)).

G. EXAMPLES

The following examples are intended to illustrate but not to limit theinvention.

Example 1 High Throughput Screening

In this screen, the standard positive control was the compound 59-0008(also denoted “OS8”), which is of the formula:

In more detail, the 2T3-BMP-2-LUC cells, a stably transformed osteoblastcell line described in Ghosh-Choudhury et al., Endocrinology (1996)137:331-39, referenced above, was employed. The cells were culturedusing α-MEM, 10% FCS with 1% penicillin/streptomycin and 1% glutamine(“plating medium”), and were split 1:5 once per week. For the assay, thecells were resuspended in a plating medium containing 4% FCS, plated inmicrotiter plates at a concentration of 5×10³ cells (in 50 μl)/well, andincubated for 24 hours at 37° C. in 5% CO₂. To initiate the assay, 50 μlof the test compound or the control in DMSO was added at 2×concentration to each well, so that the final volume was 100 μl. Thefinal serum concentration was 2% FCS, and the final DMSO concentrationwas 1%. Compound 59-0008 (10 μM) was used as a positive control.

The treated cells were incubated for 24 hours at 37° C. and 5% CO₂. Themedium was then removed, and the cells were rinsed three times with PBS.After removal of excess PBS, 25 μl of 1× cell culture lysing reagent(Promega #E153A) was added to each well and incubated for at least tenminutes. Optionally, the plates/samples could be frozen at this point.To each well was added 50 μl of luciferase substrate (Promega #E152A; 10ml Promega luciferase assay buffer per 7 mg Promega luciferase assaysubstrate). Luminescence was measured on an automated 96-wellluminometer, and was expressed as either picograms of luciferaseactivity per well or as picograms of luciferase activity per microgramof protein.

Example 2 In Vitro Bone Formation

Selected compounds and appropriate controls were assayed in vitro (exvivo) for bone formation activity (described above in “Techniques forNeonatal Mouse Calvaria Assay (in vitro)). Histomorphometricalassessments of ex vivo calvaria were carried out using an OsteoMetricsbone morphometry measurement program, according to the manufacturer'sinstructions. Measurements were determined using either a 10- or 20-foldobjective with a standard point counting eyepiece graticule.

New bone formation was determined (using a 20× objective) by measuringthe new bone area formed in one field in 3 representative sections ofeach bone (4 bones per group). Each measurement was carried out ½ fielddistance from the end of the suture. Both total bone and old bone areawere measured. Data were expressed as new bone width in mm.

Osteoblast numbers are determined by point counting. The number ofosteoblast cells lining the bone surface on both sides of the bone arecounted in one field using a 20× objective. Data are expressed asosteoblast numbers/mm of bone surface.

Alkaline phosphatase activity is measured in the incubation media asdetailed described above in “Techniques for Neonatal Mouse CalvariaAssay (in vitro)).

An example of the concept of a nitric oxide releasing bisphosphonateconjugate is seen where O-Nitroso analogue is linked to alendronate(4-amino-1-hydroxybutylidene)bis-phosphonate) (FIG. 1) to make[4-(4′-nitrooxymethyl)benzoylamino-1′-hydroxybutylidene]-bis-phosphonate(NOX-alendronate) (FIG. 2)

TABLE 1 Stimulation New Bone Formation on Neonatal Murine Calvariae withNOX-Alendronate uM New Bone Area Cell Number/ NOX-Alendronate (mm² ×10⁻³) 0.3 mm bone 0 3.5 ± 0.2  95 ± 7 0.1 3.6 ± 0.6 104 ± 8 1 4.2 ± 0.4115 ± 5 10 6.7 ± 0.7* 132 ± 5* 100 5.4 ± 0.3* 124 ± 2*

TABLE 2 Stimulation New Bone Formation on Neonatal Murine Calvariae withAlendronate uM New Bone Area Cell Number/ Alendronate (mm² × 10⁻³) 0.3mm bone 0 3.1 ± 0.3  96 ± 5 0.1 2.9 ± 0.1  98 ± 6 1.0 3.2 ± 0.5 103 ± 410 2.7 ± 0.2 101 ± 5 100 3.1 ± 0.3  97 ± 3

TABLE 3 Alkaline phosphatase activity in neonatal murine calvarialincubation media from bones treated with either NOX-Alendronate orAlendronate Alkaline Phosphatase Alkaline Phosphatase uM ODNOX-Alendronate OD Alendronate 0 0.024 + 0.006 0.027 + 0.006 0.1 0.029 +0.007 0.030 + 0.009 1 0.023 + 0.001 0.031 + 0.010 10 0.053 + 0.007*0.034 + 0.001 100 0.072 + 0.003* 0.038 + 0.003The numbers represent the mean ±standard error (n=5). *p<0.05, t-test orone-way analysis of variance (ANOVA) followed by Dunnett's test.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be incorporated within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated herein by referencefor all purposes.

Citation of the above documents is not intended as an admission that anyof the foregoing is pertinent prior art, nor does it constitute anyadmission as to the contents or date of these documents.

1. A compound comprising NO-(4-amino-1-hydroxybutylidene)bis-phosphonate(NO-alendronate), NO-(3-Amino-1-hyroxypropylidene)bis-phosphonate(NO-pamidronate), NO-(Dichloromethylene)-bis-phosphonate,(NO-clodronate),NO-[1-Hydroxy-2(3-pyridinyl)-ethylidene]-bis-phosphonate(NO-risedronate),NO-[1-Hydroxy-2-(1H-imidazole-1-yl)ethylidene]-bis-phosphonate(NO-zolondronate), NO-(1-Hydroxyethylidene)-bis-phosphonate(NO-etidronate),NO-[1-Hydroxy-3-(methylpentylamino)propylidene]-bis-phosphonate(NO-ibandronate), NO-[[(4-Chlorophenyl)thio]methylene]-bis-phosphonate(NO-tiludronate), NO-(6-Amino-1-hydroxyhexylidene)-bis-phosphonate(NO-neridronate),NO-([3-(Dimethylamino)-1-hydroxypropylidene]-bis-phosphonate(NO-olpadronate), NO-[(Cycloheptylamino)-methylene]-bis-phosphonate(NO-icadronate), orNO-[1-Hydroxy-2-imidazo-(1,2-a)pyridine-3-ylethylidene]-bis-phosphonate(NO-YH529).
 2. A pharmaceutical compound comprising the compound ofclaim 1 and a pharmaceutically acceptable carrier or excipient.
 3. Thepharmaceutical compound of claim 2, further comprising an additionalagent formulated in a single pharmaceutical formulation.
 4. Thepharmaceutical compound of claim 3, wherein the additional agentenhances bone growth or decreases bone resorption.